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Background Secreted Hedgehog (Hh) signalling molecules have profound influences on many developing and regenerating tissues. myoblasts respond differently NLG919 to Shh: in some slow myosin expression is usually increased whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells yet simultaneously NLG919 enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres however do not express extra slow myosin. Conversely Hh loss of function in the limb bud caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody reduces early slow muscle formation and differentiation but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis. Conclusions Taken together the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires NLG919 Hh for normal differentiation and slow myosin expression. We propose a simple model of how direct and indirect effects of Hh regulate early limb myogenesis. Background Each muscle in a developing chick limb acquires a unique character from its inception [1]. Fibres form by the terminal differentiation of dividing myoblasts that elongate in particular orientations to form specific attachments to the skeleton. Simultaneously the fibres of each muscle take on gene expression patterns characteristic of their future function. For example those muscles destined to maintain body posture express certain isoforms of slow myosin from their inception whereas future fast muscle regions fail to express this slow myosin [2]. It has been suggested that distinct cell lineages underlie the formation of slow and fast muscle fibres and much evidence for myoblast heterogeneity has been obtained from studies both in vitro and in vivo [[3-7] Prkd1 reviewed in [8]]. Nevertheless it is usually clear that for fibres to undergo differentiation NLG919 at the appropriate time and place extrinsic cues must regulate muscle patterning. Work on muscle patterning in somites over the past decade has shown that various protein factors secreted by adjacent tissues act as extrinsic signals regulating the formation and fate of myogenic cells [[9] reviewed in [10-12]]. One such factor is usually Sonic hedgehog (Shh) derived from the ventral midline which is required for expression of markers of the earliest populace of myogenic cells in the medial somite of both birds and mice [13-15]. These medial somitic cells contribute to the early-born muscle fibres of the myotome but their subsequent fate is not known in amniotes [16 17 Ventral midline Hedgehog (Hh) signals are also required for formation of the earliest muscle cells in the zebrafish embryo the adaxial slow cells [[18 19 reviewed in [20]]. The fate of these cells is known they generate a populace of slow muscle fibres that migrate to form a layer of slow muscle that covers the lateral surface of the somite [21 22 In all vertebrates examined a second myogenic cell populace arises in the lateral somite by a distinct Hh-independent genetic pathway in response to signals from more lateral and dorsal tissues. Signals such as FGFs BMPs and WNTs and their antagonists are primary candidates for patterning of lateral somitic cells at least in amniotes [reviewed in [8 9 23 24 Wnt proteins from dorsal tissues are also implicated in medial myogenesis [25-30]. In the somite induction of precursor myoblast populations is occurring close in space and time to terminal differentiation of myoblasts into contractile fibres. This makes analysis of the precise effects of extrinsic signals hard to determine. For example Shh can promote both primary myogenesis and subsequent cell survival in somitic explants and in vivo but the precise target cell populations are unclear [13 15 31 In contrast in the limb bud myogenic induction and terminal differentiation are.